Smirnova I V, Zhang S X, Citron B A, Arnold P M, Festoff B W
Neurobiology Research Laboratory (151R), Department of Veterans Affairs Medical Center, Kansas City, Missouri 64128, USA.
J Neurobiol. 1998 Jul;36(1):64-80.
Apoptosis, often also termed "programmed cell death", occurs in normal development in the brain and spinal cord. Important to concepts of disease and potential intervention is the exciting finding that apoptosis is also found after neurotrauma and in a number of neurodegenerative diseases. Although the precise mechanism of neuronal cell loss remains unknown, much emphasis has been placed recently on the activation of cell death protease cascades within the cell. How these cascades may be activated, especially from extracellular influences, is currently poorly understood. Thrombin, the multifunctional coagulation protease, is an early phase modulator at sites of tissue injury and has been shown to induce cell death in neurons by an apoptotic mechanism by activating its receptor, PAR-1. Using a model motor neuronal cell line, NSC19, which we have shown undergoes apoptosis after treatment with classic apoptosis inducers such as the topoisomerase inhibitors camptothecin and etoposide, we unambiguously found that nanomolar thrombin induced characteristic signs of apoptosis. Strikingly, endonucleolysis was accompanied by an increase in caspase-3-like activity in cellular extracts, which correlated with both detection of caspase-induced signature cleavage of the cortical cytoskeleton component nonerythroid spectrin (alpha-fodrin) and identification of increased accessibility of a caspase cleavage domain, using an antibody (Ab127) made against a synthetic peptide KGDEVD. Demonstrating that thrombin activation of death proteases was linked to cell death, we were able to inhibit thrombin-induced apoptosis by using a caspase family inhibitor, benzyloxycarbonyl-Asp-(oMe)-fluoromethyl ketone (Boc-D-FMK). These novel results demonstrate that thrombin serves as an extracellular "death signal" to activate intracellular protease pathways. These pathways lead to apoptotic cell death and can be modulated by inhibiting caspase activity downstream to PAR-1.
凋亡，通常也被称为“程序性细胞死亡”，发生于大脑和脊髓的正常发育过程中。对于疾病概念和潜在干预措施而言，一个令人兴奋的发现是，在神经创伤后以及多种神经退行性疾病中也能发现凋亡现象。尽管神经元细胞死亡的确切机制仍不明确，但最近人们十分关注细胞内细胞死亡蛋白酶级联反应的激活。目前，对于这些级联反应如何被激活，尤其是受到细胞外影响时的激活方式，了解甚少。凝血酶是一种多功能凝血蛋白酶，是组织损伤部位的早期调节剂，已被证明可通过激活其受体PAR - 1，以凋亡机制诱导神经元细胞死亡。我们使用一种运动神经元细胞系NSC19，该细胞系在接受拓扑异构酶抑制剂喜树碱和依托泊苷等经典凋亡诱导剂处理后会发生凋亡，我们明确发现纳摩尔浓度的凝血酶可诱导凋亡的特征性迹象。引人注目的是，核酸内切作用伴随着细胞提取物中caspase - 3样活性的增加，这与检测到caspase诱导的皮质细胞骨架成分非红细胞血影蛋白（α - fodrin）的标志性裂解以及使用针对合成肽KGDEVD制备的抗体（Ab127）鉴定出caspase裂解结构域可及性增加相关。为了证明凝血酶对死亡蛋白酶的激活与细胞死亡有关，我们能够使用caspase家族抑制剂苄氧羰基 - Asp - （oMe） - 氟甲基酮（Boc - D - FMK）抑制凝血酶诱导的凋亡。这些新结果表明，凝血酶作为一种细胞外“死亡信号”来激活细胞内蛋白酶途径。这些途径导致凋亡性细胞死亡，并且可以通过抑制PAR - 1下游的caspase活性来调节。
